The goal of this project is to define the molecular mechanisms involved in the replication of HIV and related retroviruses and to develop new strategies for AIDS therapy. Our research is currently focused on two broad areas of interest: reverse transcription and virus assembly. During reverse transcription, there are two strand transfer events that are required for synthesis of full-length plus- and minus-strand DNA copies of the viral RNA genome. Previously we demonstrated that in both cases, the activity of the HIV-1 nucleocapsid protein (NC) is required to achieve efficient and specific viral DNA synthesis. HIV-1 NC has two zinc fingers, each containing the invariant metal ion binding residues CCHC. Although it has been known for many years that the zinc fingers are required for viral RNA packaging as well as for other functions in virus replication, efforts to identify these additional activities have been hampered by the lack of definitive in vitro assays. Using a mutant in which zinc coordination is severely disrupted by changing the six cysteine residues to serine, we have now established that the NC CCHC motifs are essential for efficient minus- and plus-strand transfer and contribute to destabilization of strong secondary structures in nucleic acid strand transfer intermediates. The importance of zinc finger interactions for NC activity in reverse transcription indicates that drugs which target the zinc finger structures would provide a new approach for effective anti-HIV therapy. In other work, NMR studies have shown that the HIV-1 18-nt minus-strand primer binding site [(-) PBS] DNA, which anneals to the complementary DNA during plus-strand transfer, forms a relatively stable, small stem-loop structure. The broadening and reduction of signals from Watson-Crick imino protons when HIV-1 NC is bound to (-) PBS DNA provides the first direct evidence that NC destabilizes local nucleic acid structure. For studies on the role of the HIV-1 capsid protein (CA) in virus replication, we have made several viral mutants with alanine substitutions in CA residues. Virus particles are produced, but are non-infectious, exhibit aberrant core morphology, and are unable to initiate viral DNA synthesis in the infected cell. Taken together, these results reveal the close connection between proper core morphology and the ability to undergo reverse transcription. Analysis of additional alanine-substitution CA mutants is now in progress.